EP3118331A1 - Oligonucléotide simple bicatéaire partiellement neutre - Google Patents

Oligonucléotide simple bicatéaire partiellement neutre Download PDF

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Publication number
EP3118331A1
EP3118331A1 EP16179734.5A EP16179734A EP3118331A1 EP 3118331 A1 EP3118331 A1 EP 3118331A1 EP 16179734 A EP16179734 A EP 16179734A EP 3118331 A1 EP3118331 A1 EP 3118331A1
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Prior art keywords
detection
stranded oligonucleotide
detection unit
nucleotide
unit according
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German (de)
English (en)
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Chan Hardy Wai-Hong
Yang Yuh-Shyong
Chen Wen-Yih
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HELIOS BIOELECTRONICS INC.
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Orizhan Bioscience Ltd
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Definitions

  • This invention relates to a molecular detection technique. More particularly, the invention relates to a partially neutral single-stranded oligonucleotide.
  • Molecular detection plays an important role in clinical diagnosis and molecular biology research.
  • Several systems have been developed to perform molecular detection for detecting and/or identifying a target biomolecule in a sample.
  • single-stranded oligonucleotides are used in the detection and/or identification of a target with specific nucleotide sequence based on Watson-Crick base-pairing rules.
  • the detection and/or identification of the target usually involves the ability to distinguish a single base variant.
  • several single-stranded oligonucleotides are coated on one microarray substrate for hybridizing a fluorescence-labeled sample mixture.
  • the operation condition such as temperature for hybridization of each single-stranded oligonucleotide in each detection and/or identification must be accommodated.
  • the sensitivity and specificity is critical to the microarray avoiding any false signal.
  • the melting temperature is defined as the temperature at which half of the oligonucleotide strands are in the random coil or single-stranded state.
  • the melting temperature depends on the length of the oligonucleotide and its specific nucleotide sequence.
  • PNA peptide nucleic acid
  • Science 254 (5037): 1497-500 a peptide nucleic acid (PNA) has been disclosed ( Nielsen PE, Egholm M, Berg RH, Buchardt O, 1991. "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide.” Science 254 (5037): 1497-500 ), and the backbone of PNA comprises repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • a morpholino also known as a morpholino oligomer and as a phosphorodiamidate morpholino oligomer (PMO) has also been developed ( Summerton, J; Weller D, 1997. "Morpholino Antisense Oligomers: Design, Preparation and Properties.” Antisense & Nucleic Acid Drug Development 7 (3): 187-95 ).
  • the structure of PMO has a backbone of methylenemorpholine rings and phosphorodiamidate linkages. Because of completely unnatural backbones, PMO cannot not be recognized by cellular proteins such as enzymes, and it restricts the applications involved in protein binding as well as enzyme recognition.
  • a locked nucleic acid is provided as a modified RNA nucleotide ( Satoshi Obika; Daishu Nanbu; Yoshiyiki Hari; Ken-ichiro Morio; Yasuko In; Toshimasa Ishida; Takeshi Imanishi, 1997. "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3'-endo sugar puckering.” Tetrahedron Lett. 38 (50): 8735-8 ). The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon.
  • the bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes.
  • the locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the melting temperature of oligonucleotides.
  • the popularity of LNA is limited by its chemistry of synthesis and the price of having LNA designed and synthesis.
  • US 2014/0235465 A1 discloses a neutralized DNA (nDNA). All of the charged oxygen ions (O - ) on the phosphate backbone have been alkylated, so that the backbone is totally electrically neutral. This modification increases the hybridization efficiency between complementary single stranded DNAs and minimizes the salt required for hybridization, which results in higher sensitivity of the detection.
  • nDNA applied to hybridization does not produce satisfactory specificity, and nonspecific hybridization occurs in salt concentrations for regular DNA hybridization conditions.
  • a field-effect transistor has been employed to detect and/or identify a target oligonucleotide as well as an antibody-antigen binding, both of which benefit from the absence of labeling requirements for reagents and the ready availability of commercial manufacturing sources for FET sensors.
  • Sensitivity of the FET sensor is highly dependent on detection distance (debye length) between the transistor surface and the actual detected molecules. Most current types of FET are less than satisfactory as gene detection devices in terms of sensitivity. This is mainly due to the requirement of relatively high salt concentrations for DNA/DNA or DNA/RNA hybridizations. Hybridization of highly charged biomolecules requires an appropriate ionic strength to suppress the charge repulsive forces.
  • ions in the hybridization buffer also reduce the FET debye length and hence diminish detection sensitivity.
  • the large size of the antibody as compared to other biomolecules also reduces the detection sensitivity of the FET.
  • the surface charges distribution of antibody and the binding orientation of the bound antibody make FET detection difficulty to be resolved and be quantitatively analyzed.
  • medium concentrations of salt in the binding buffer also reduce the debye length and, in turn, lower the detection sensitivity as well.
  • the invention is to provide a partially neutral single-stranded oligonucleotide comprising at least one electrically neutral nucleotide and at least one negatively charged nucleotide.
  • the present invention is also to provide a detection unit comprising the partially neutral single-stranded oligonucleotide as mentioned above.
  • the present invention is also to provide a detection system comprising a plurality of the detection units as mentioned above.
  • the present invention is also to provide a method of detection comprising performing the detection with the detection unit as mentioned above.
  • the invention is to provide a partially neutral single-stranded oligonucleotide comprising at least one electrically neutral nucleotide and at least one negatively charged nucleotide.
  • oligonucleotide refers to a nucleotide oligomer.
  • nucleotide refers to an organic molecule composed of a nitrogenous base, a sugar, and one or more phosphate groups; preferably one phosphate group.
  • the nitrogenous base includes a derivative of purine or pyrimidine.
  • the purine includes substituted or unsubstituted adenine and substituted or unsubstituted guanine; the pyrimidine includes substituted or unsubstituted thymine, substituted or unsubstituted cytosine and substituted or unsubstituted uracil.
  • the sugar is preferably a five-carbon sugar, more preferably substituted or unsubstituted ribose or substituted or unsubstituted deoxyribose.
  • the phosphate groups form bonds with the 2, 3, or 5-carbon of the sugar; preferably, with the 5-carbon site.
  • the sugar of one nucleotide is joined to the adjacent sugar by a phosphodiester bridge.
  • a proper form of the partially neutral single-stranded oligonucleotide can be chosen as needed based on the disclosure of the invention.
  • the partially neutral single-stranded oligonucleotide is DNA or RNA; more preferably DNA.
  • the partially neutral single-stranded oligonucleotide according to the invention comprises at least one electrically neutral nucleotide and at least one negatively charged nucleotide.
  • the manner of rendering a nucleotide electrically neutral is not limited.
  • the electrically neutral nucleotide comprises a phosphate group substituted by an alkyl group.
  • the alkyl group is a C 1 -C 6 alkyl group; more preferably, the alkyl group is a C 1 -C 3 alkyl group. Examples of the C 1 -C 3 alkyl group include but are not limited to methyl, ethyl and propyl.
  • FIG. 1 shows one preferred embodiment of the electrically neutral nucleotide according to the invention.
  • the negatively-charged oxygen atom in the phosphate group is changed to a neutral atom without charge.
  • the way to substitute the phosphate group with the alkyl group can be applied according to common chemical reactions.
  • the negatively charged nucleotide according to the invention comprises a phosphate group with at least one negative charge.
  • the unmodified nucleotide is preferably a naturally occurring nucleotide without modification or substitution.
  • the negatively charged nucleotide comprises an unsubstituted phosphate group.
  • the partially neutral single-stranded oligonucleotide according to the invention is partially rendered electrically neutral.
  • the sequence or length is not limited, and the sequence or length of the partially neutral single-stranded oligonucleotide can be designed according to a target molecule based on the disclosure of the invention.
  • the number of the electrically neutral nucleotides and negatively charged nucleotides depend on the sequence of the partially neutral single-stranded oligonucleotide and the condition under which a reaction is to be detected.
  • the positions of the electrically neutral nucleotide and negatively charged nucleotide also depend on the sequence of the partially neutral single-stranded oligonucleotide and the condition under which a reaction is to be detected.
  • the number and position of the electrically neutral nucleotides and negatively charged nucleotides can be designed according to the available information based on the disclosure of the invention.
  • the number and position of the electrically neutral nucleotides in any probes can be designed by molecular modeling calculation based on double stranded (ds) structural energy, and the melting temperature (Tm) of dsDNA/DNA or dsDNA/RNA can then be determined by reference to the structural energy.
  • ds double stranded
  • Tm melting temperature
  • the partially neutral single-stranded oligonucleotide comprises a plurality of the electrically neutral nucleotides, and at least one negatively charged nucleotide is positioned between two of the electrically neutral nucleotides; more preferably, at least two negatively charged nucleotides are positioned between two of the electrically neutral nucleotides.
  • At least one electrically neutral nucleotide when applying the partially neutral single-stranded oligonucleotide according to the invention in the detection and/or identification of a single base variant, at least one electrically neutral nucleotide is positioned near the single base variant; more preferably, 2, 3, 4, or 5 electrically neutral nucleotides are positioned near the single base variant. In another aspect, at least one electrically neutral nucleotide is positioned at the downstream or upstream site of the single base variant; preferably, at least one electrically neutral nucleotide is positioned at the downstream and upstream sites of the single base variant.
  • the melting temperature difference between perfect match double-stranded oligonucleotides and mismatched double-stranded oligonucleotides of the partially neutral single-stranded oligonucleotide according to the invention is higher compared with that of a conventional DNA probe.
  • the electrostatic repulsion force between two strands is lowered by introducing the neutral oligonucleotide, and the melting temperature is raised thereby.
  • the melting temperature difference is adjusted to a desired point, providing a better working temperature or temperature range to differentiate the perfect and mismatched oligonucleotides, thereby improving the detection specificity.
  • Such design benefits the consistency of the melting temperature of different partially neutral single-stranded oligonucleotides integrated in one chip or array.
  • the number of reactions to be detected can be raised dramatically with high specificity and more detection units can be incorporated into a single detection system.
  • the design provides better microarray operation conditions.
  • the present invention is also to provide a detection unit comprising the partially neutral single-stranded oligonucleotide as mentioned above.
  • the term "detection” refers to a process of discovering or determining the existence or presence of a target molecule, and preferably, a process of identifying the target molecule.
  • the detection comprises quantifying the target molecule in a sample.
  • a reaction applied in the detection includes but is not limited to hybridization between oligonucleotide molecules, protein-protein interaction, receptor-ligand binding, oligonucleotide-protein interaction, polysaccharide-protein interaction, or small molecule-protein interaction.
  • the present invention is also to provide a detection system comprising a plurality of the detection units as mentioned above.
  • the term "unit" refers to a component for carrying out the reaction applied in the detection.
  • the unit is for carrying out a single reaction.
  • several units are contained in one system to carry out several detections in one manipulation.
  • a plurality of detection units may be incorporated in a detection system.
  • the detection system is a microarray or a chip.
  • the term "molecule" refers to a small molecule or a macromolecule.
  • the molecule is a macromolecule such as a protein, peptide, nucleotide, oligonucleotide or polynucleotide.
  • the molecule is naturally occurring or artificial.
  • the molecule is purified or mixed with other contents.
  • the expression pattern of the molecule is different in a normal condition and in an abnormal condition, such as a disease.
  • the expression pattern of the molecule is different in different cell types.
  • the molecules are DNA molecules, RNA molecules, antibodies, antigens, enzymes, substrates, ligands, receptors, cell membrane-associated proteins or cell surface markers.
  • the DNA molecules are preferably a gene or an untranscripted region.
  • the RNA molecules are preferably mRNA, micro RNA, long untranslated RNA, rRNA, tRNA, or siRNA.
  • target molecule refers to a specified molecule to be detected or identified from a pool of molecules.
  • the sample according to the invention is derived from a naturally occurring origin or derived from an artificial manipulation.
  • the sample is derived from a naturally occurring origin such as an extract, body fluid, tissue biopsy, liquid biopsy, cell culture.
  • the sample is processed according to the reaction required for detection. For example, the pH value or ion strength of the sample may be adjusted.
  • the partially neutral single-stranded oligonucleotide according to the invention in the detection unit may be presented in a solution or linked to a support.
  • the detection unit further comprises a solid surface; and the partially neutral single-stranded oligonucleotide is attached on or located near the solid surface.
  • solid surface refers to a solid support including but not limited to a polymer, paper, fabric, or glass.
  • the solid surface to be employed varies depending on the reaction signal to be detected. For example, when the detection adopts a field-effect transistor to monitor the signal, the solid surface is a transistor surface of the field-effect transistor; when the detection adopts a surface plasmon resonance, the solid surface is a metal surface of a surface plasmon resonance; when the detection adopts a microarray detection system, the solid surface is a substrate surface of the microarray.
  • the material of the solid surface is silicon; preferably polycrystalline silicon or single crystalline silicon; more preferably polycrystalline silicon.
  • Polycrystalline silicon is cheaper than single crystalline silicon, but because the polycrystalline has more grain boundary, a defect usually occurs in the grain boundary that hinders electron transduction. Such phenomenon makes the solid surface uneven and quantification difficult. Furthermore, ions may penetrate into the grain boundary of the polycrystalline and cause detection failure in solution. In addition, polycrystalline silicon is not stable in air. The abovementioned drawbacks, however, would not interfere with the function of the detection unit according to the invention.
  • the detection unit further comprises a signal detection component.
  • the signal detection component is applied for detecting whether hybridization and/or binding occurs between the partially neutral single-stranded oligonucleotide and the target molecule.
  • the detection component is a field-effect transistor, a surface plasmon resonance, a microscope, a spectrometer, an electrophoresis device, or an electrochemical sensor. In one embodiment of the invention, referring to FIG.
  • the charge changes compared to that of the partially neutral single-stranded oligonucleotide because the target molecule of DNA or RNA carries abundant negative charges. If a mismatch occurs between the partially neutral single-stranded oligonucleotide and the target molecule such as single nucleotide polymorphism detection, no complex forms.
  • the signal detection component is used for detecting the charge change and monitoring if hybridization occurs.
  • the signal detection component is usually integrated with the solid surface for detecting the voltage of the solid surface. Artisans skilled in this field are able to design the detection component according to the invention.
  • the partially neutral single-stranded oligonucleotide comprises a first portion attached to the solid surface; the length of the first portion is about 50% of the total length of the partially neutral single-stranded oligonucleotide; and the first portion comprises at least one electrically neutral nucleotide and at least one negatively charged nucleotide; more preferably, the length of the first portion is about 40% of the total length of the partially neutral single-stranded oligonucleotide; still more preferably, the length of the first portion is about 30% of the total length of the partially neutral single-stranded oligonucleotide.
  • the partially single-stranded nucleotide further comprises a second portion adjacent to the first portion.
  • the second portion is located in the distal end relative to the solid surface.
  • the second portion comprises at least one electrically neutral nucleotide and at least one negatively charged nucleotide.
  • the description of the electrically neutral nucleotide and the negatively charged nucleotide is the same as that of the first portion and is not repeated herein.
  • the manner of attaching the partially neutral single-stranded oligonucleotide and the solid surface depends on the material of the solid surface and the type of the partially neutral single-stranded oligonucleotide.
  • the partially neutral single-stranded oligonucleotide links to the solid surface through a covalent bond.
  • the covalent bond include but are not limited to the following methods, depending on the solid surface chemistry and the modification of the oligonucleotide.
  • the solid surface is modified by using (3-Aminopropyl)triethoxysilane (APTES).
  • the silicon atom in the molecule of APTES performs a covalent bond with the oxygen atom of the hydroxyl group and it converts the surface's silanol groups (SiOH) to amines; then the 5'-amino group of partially neutral single-stranded oligonucleotide is covalently bonded with the solid surface amines group by glutaraldehyde ( Roey Elnathan, Moria Kwiat, Alexander Pevzner, Yoni Engel, Larisa Burstein Artium Khatchtourints, Amir Lichtenstein, Raisa Kantaev, and Fernando Patolsky, Biorecognition Layer Engineering: Overcoming Screening Limitations of Nanowire-Based FET Devices, Nano letters, 2012, 12, 5245-5254 ).
  • glutaraldehyde Roey Elnathan, Moria Kwiat, Alexander Pevzner, Yoni Engel, Larisa Burstein Artium Khatchtourints,
  • the solid surface is modified into self-assembly monolayer molecules with different functional groups for covalently linking to different functional groups of the partially neutral single-stranded oligonucleotide by various chemical reactions ( Srivatsa Venkatasubbarao, Microarrays - status and prospects, TRENDS in Biotechnology Vol.22 No. 12 December 2004 ; Ki Su Kim, Hyun-Seung Lee, Jeong-A Yang, Moon-Ho Jo and Sei Kwang Hahn, The fabrication, characterization and application of aptamer-functionalized Si-nanowire FET biosensors, Nanotechnology 20 (2009 )).
  • the partially neutral single-stranded oligonucleotide is located near the solid surface.
  • the partially neutral single-stranded oligonucleotide is not necessary to directly bind to the solid surface, provided that the distance between the partially neutral single-stranded oligonucleotide and the solid surface is short enough allowing the detection component to detect the voltage change.
  • the distance between the solid surface and the partially neutral single-stranded oligonucleotide is about 0 to about 10 nm; more preferably about 0 to about 5 nm.
  • the detection unit according to the invention further comprises a signal amplifier.
  • the signal amplifier according to the invention preferably refers to a component that enhances the detection of the voltage change of the solid surface.
  • the signal amplifier is nanogold particles attached to one end of the partially neutral single-stranded oligonucleotide.
  • the detection unit further comprises a buffer with an ionic strength lower than about 50 mM; more preferably lower than about 40 mM, 30 mM, 20 mM or 10 mM.
  • a buffer with an ionic strength lower than about 50 mM; more preferably lower than about 40 mM, 30 mM, 20 mM or 10 mM.
  • the detection unit further comprises a biomolecule for expanding the application of the detection unit.
  • a biomolecule for expanding the application of the detection unit.
  • the biomolecule include but are not limited to a single-stranded DNA molecule, a single-stranded RNA molecule, a polypeptide or a protein.
  • the detection unit further comprises a protein linked to the partially neutral single-stranded oligonucleotide.
  • a protein linked to the partially neutral single-stranded oligonucleotide Artisans skilled in this field can complete the linkage between the protein and the partially neutral single-stranded oligonucleotide, for example, by specific affinity binding.
  • the detection system serves as a protein chip with improved hybridization specificity for detecting a target interaction with the linked protein.
  • the detection unit further comprises a single-stranded DNA as a normal probe, and the partially neutral single-stranded oligonucleotide serves as a recovery probe.
  • the single-stranded DNA as the normal probe may be a modified signal-stranded DNA or an unmodified single-stranded DNA; preferably, a partially neutral single-stranded DNA.
  • the recovery probe and the normal probe both have binding affinity to a target; more preferably, the recovery probe has higher affinity to the target than the normal probe.
  • the recovery probe competes with the normal probe in a complex formed with the normal probe and the target. Such a displacement can be readily observed, for example, with the field-effect transistor.
  • the normal probe is designed to detect the presence of the target, and the recovery probe is design to detect the presence of single nucleotide polymorphism with a mismatched nucleotide.
  • the normal probe is first to form a complex with the target, and the recovery probe is then bound to the target in the complex. The signal is monitored to detect if a mismatch is present between the recovery probe and the target.
  • the recovery probe links to nanogold particles for amplifying the signal.
  • the present invention is also to provide a method of detection comprising performing the detection with the detection unit as mentioned above.
  • the detection includes but is not limited to non-labeling gene expression, polymerase chain reaction, in situ hybridization, single nucleotide polymorphism (SNP) detection, RNA detection, DNA conjugated protein and protein detection.
  • SNP single nucleotide polymorphism
  • the detection unit by applying the detection unit according to the invention, a very small amount of the target molecule can be detected in the sample without amplifying the target molecule in a polymerase chain reaction.
  • the detection preferably comprises detecting a target of less than 10 -9 Molar in the sample, and the method is free of a polymerase chain reaction.
  • the improved hybridization specificity in gene detection can be seen mainly in two aspects of FET detection compared to a conventional detection.
  • the partially neutral single-stranded oligonucleotide designed on the field effect transistor microarray or surface plasmon resonance microarray provides quantitative gene expression, protein and SNP detection in a non-labelled and a high throughput fashion.
  • Deoxy cytidine (n-ac) p-methoxy phosphoramidite, thymidine p-methoxy phosphoramidite, deoxy guanosine (n-ibu) p-methoxy phosphoramidite, and deoxy adenosine (n-bz) p-methoxy phosphoramidite were used to synthesize an oligonucleotide according to a given sequence based on solid-phase phosphotriester synthesis or by Applied Biosystems 3900 High Throughput DNA Synthesizer (provided by Genomics® Biosci & Tech or Mission Biotech).
  • the synthesized oligonucleotide was reacted with weak alkaline in toluene at room temperature for 24 hours, and the sample was subjected to ion-exchange chromatography to adjust the pH value to 7. After the sample was concentrated and dried, the partially neutral single-stranded oligonucleotide was obtained.
  • Example 2 Partially neutral single-stranded oligonucleotide applied in FET detection Complementary H1:
  • Probe immobilization was performed by functionalization of the SiNW surface layer (SiO 2 ).
  • APTES (3-Aminopropyl)triethoxysilane
  • the silicon atom in the molecule of APTES performed a covalent bond with the oxygen of the hydroxyl group and converted the surface's silanol groups (SiOH) to amines.
  • Samples were immersed in 2% APTES (99% EtOH) for 30 minutes and then heated to 120°C for 10 min. After this step, amino groups (NH 2 ) were the terminal units from the surface.
  • glutaraldehyde was used as a grafting agent for DNA immobilization.
  • Glutaraldehyde binding was achieved through its aldehyde group (COH) to ensure a covalent bond with the amino group of APTES.
  • COH aldehyde group
  • samples were immersed in 12.5% glutaraldehyde (10mM sodium phosphate buffer) in liquid for 1 hour at room temperature.
  • probe immobilization 5'-amino group of DNA strands were linked to the aldehyde groups of the linker.
  • a 500 ⁇ L drop solution of 1 ⁇ mol DNA probes was deposited onto the NWs for 18 hours.
  • PDMS polydimethylsiloxane fluidic system
  • PDMS polydimethylsiloxane fluidic system
  • Complementary and non-complementary targets were used, with various concentrations with dilution with bis-tris propane [1,3-bis(tris(hydroxymethyl)methylamino)propane] solution.
  • samples were washed with bis-tris buffer for 10 min to remove excess targets.
  • Keithley 2400 was used to detect the NWFET electrical characteristics (Id vs. Vg curves).
  • nDNA and partially neutral single-stranded DNA in low salt concentrations have higher FET sensitivity in ⁇ V of DNA in high salt concentration.
  • Partially neutral single-stranded DNA with 3' end modifications has the highest FET sensitivity.
  • nDNA and partially neutral single-stranded DNA probes have higher concentration sensitivity (slope of V. vs. Conc.), especially in low target concentration ranges.
  • the results of detecting ultralow target concentrations at 1 mM bis-tris are shown in FIG 7 .
  • the target concentration can be detected as low as 0.1 fM with partially neutral single-stranded DNA probe on FET.
  • Example 3 Partially neutral single-stranded oligonucleotide applied in polymerase chain reaction
  • the partially neutral single-stranded oligonucleotides are applied in polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR) of SYBRTM Green system.
  • FIGs. 8A and 8B The results of PCR are shown in FIGs. 8A and 8B . Comparing the results of using DNA and those of using the partially neutral single-stranded oligonucleotides ( FIG. 8 A: Regular DNA primer (FP-R and RP-R of Table 2) vs. neutralized DNA modifications primer (FP-n 10, 14 of Table 2) ; FIG. 8 B: Regular DNA primer(FP-R and RP-R of Table 2) vs. neutralized DNA modifications primer (FP-n 11, 13 of Table 2)), the primer specificity of the partially neutral single-stranded oligonucleotides is significantly improved.
  • FIG. 8 A Regular DNA primer (FP-R and RP-R of Table 2) vs. neutralized DNA modifications primer (FP-n 10, 14 of Table 2)
  • FIG. 8 B Regular DNA primer(FP-R and RP-R of Table 2) vs. neutralized DNA modifications primer (FP-n 11, 13 of Table 2)
  • Example 4 Partially neutral single-stranded oligonucleotide applied in detecting miRNA by in situ hybridization
  • ISHyb in situ hybridization were performed the manufacture's instructions of ISHyb in situ hybridization kit (BioChain, Newark, CA, USA).
  • the test cells were immobilized with 4% paraformaldehyde (DEPC-PBS) for 20 minutes and then washed with DEPC-PBS three times (5 minutes each time).
  • the test cells were treated with proteinase (10 ⁇ g/ml) for 8 minutes and washed with DEPC-PBS for 5 minutes.
  • the cells were further immobilized with 4% PFA (DEPC-PBS) for 15 minutes then washed with DEPC-PBS three times (5 minutes each time).
  • the washed test cells were treated with prehybridization solution (BioChain, Newark, CA, USA) at 65°C for 4 hours.
  • NBT/BCIP nitro blue tetrazolium/5-bromo-4-chloro-3indolyl phosphate
  • NBT + 3.3 ⁇ l BCIP nitro blue tetrazolium/5-bromo-4-chloro-3indolyl phosphate
  • Example 5 Partially neutral single-stranded oligonucleotide applied in detecting miRNA
  • Target miRNA miR107 (1pM): AGCAGCAUUGUACAGGGCUAUCA (SEQ ID No. 19) miR579-3p (1pM): UUCAUUUGGUAUAAACCGCGAUU (SEQ ID No. 20) miR885-5p (1pM): UCCAUUACACUACCCUGCCUCU (SEQ ID No. 21) Probes are shown in Table 6. Table 6: Name Sequences (5' to 3') SEQ ID No.
  • probe 107 (DNA) NH 2 -C6-TGATAGCCCTGTACAATGCTGCT 22 probe 107 (modified2) NH 2 -C6-T n GAT n AGC n CCT n GTACAATGCTGCT 23 probe 579-3p (DNA) NH 2 -C6-AATCGCGGTTTATACCAAATGAA 24 probe 579-3p (modified2) NH 2 -C6-A n ATC n GCG n GTT n TATACCAAATGAA 25 probe 885-5p (DNA) NH 2 -C6-AGAGGCAGGGTAGTGTAATGGA 26 probe 885-5p (modified2) NH 2 -C6-A n GAG n GCA n GGG n TAGTGTAATGGA 27
  • the surface modification for probe immobilization was as described in Example 2.
  • One 1pM miRNA (complementary to the probe) in bis-tris propane buffer was added to the probe-immobilized chip for reacting for 30 minutes.
  • the chip was washed with bis-tris propane buffer for 10 minutes. The signal was monitored as described in Example 2.
  • the results are shown in FIG. 14 .
  • the ⁇ V/mV of partially single-stranded oligonucleotide probes is higher than that of DNA probe.
  • the partially neutral single-stranded probe has higher sensitivity.

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EP3118330B1 (fr) 2019-09-11
JP6998574B2 (ja) 2022-02-04
EP3118330A1 (fr) 2017-01-18

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